Functional Roles of Poleward Microtubule Flux During Mitosis

Poleward microtubule flux is a conserved process during mitosis and meiosis in metazoan cells and is defined as the translocation of spindle microtubules toward spindle poles coupled to the depolymerization of their minus-ends. In some cell types, the rate of poleward microtubule flux matches that of poleward chromatid movement during anaphase A, suggesting that it pulls chromatids poleward. However, in other cell types, the rate of poleward microtubule flux is significantly slower than chromatid movement during anaphase A, suggesting that it makes little contribution to chromatid movement. This discrepancy led to speculation that flux is maintained in these cells to fulfill other functional roles aside from contributing to anaphase A chromatid movement. These roles include contributing to chromosome alignment, regulating spindle size and microtubule turnover, and correcting errors in chromosome attachment to spindle microtubules. Here, we discuss recent data that begin to pinpoint the functional roles of poleward microtubule flux during mitosis and meiosis.     

Poleward microtubule flux mitotic spindles assembled in vitro

In the preceding paper we described pathways of mitotic spindle assembly in cell-free extracts prepared from eggs of Xenopus laevis. Here we demonstrate the poleward flux of microtubules in spindles assembled in vitro, using a photoactivatable fluorescein covalently coupled to tubulin and multi-channel fluorescence videomicroscopy. After local photoactivation of fluorescence by UV microbeam, we observed poleward movement of fluorescein-marked microtubules at a rate of 3 microns/min, similar to rates of chromosome movement and spindle elongation during prometaphase and anaphase. This movement could be blocked by the addition of millimolar AMP-PNP but was not affected by concentrations of vanadate up to 150 microM, suggesting that poleward flux may be driven by a microtubule motor similar to kinesin. In contrast to previous results obtained in vivo (Mitchison, T. J. 1989. J. Cell Biol. 109:637-652), poleward flux in vitro appears to occur independently of kinetochores or kinetochore microtubules, and therefore may be a general property of relatively stable microtubules within the spindle. We find that microtubules moving towards poles are dynamic structures, and we have estimated the average half-life of fluxing microtubules in vitro to be between approximately 75 and 100 s. We discuss these results with regard to the function of poleward flux in spindle movements in anaphase and prometaphase.     

Cell division and the microtubular cytoskeleton]

Kinetochore microtubules result from an interaction between astral microtubules and the kinetochore of the chromosomes after breakdown of the nuclear envelope at the end of prophase. In this process, the end of a microtubule projecting from one of the polar regions contacts the primary constriction of a chromosome. The latter then undergoes rapid poleward movement. Concerning the mechanism of anaphase chromosome movement, the motive force for the chromosome-to-pole movement appears to be generated at the kinetochore or in the region very close to it. It has not been determined whether chromosomes propel themselves along stationary kinetochore microtubules by a motor at the kinetochore, or they are pulled poleward by a traction fiber consisting of kinetochore microtubules and associated motors. As chromosomes move poleward coordinate disassembly of kinetochore microtubules might occur from their kinetochore ends. In diatom and yeast spindles, elongation of the spindle in anaphase (anaphase B) may be explained by microtubule assembly at polar microtubule ends in the spindle mid-zone and sliding of the antiparallel microtubules from the opposite poles. The sliding force appears to be generated through an ATP-dependent microtubule motor. In isolated sea urchin spindles, the microtubule assembly at the equator alone might provide the force for spindle elongation, although, in addition, involvement of microtubule sliding by a GTP-requiring mechanochemical enzyme cannot be excluded. Discussions were made on possible participation in anaphase chromosome movement of such microtubule motors as dynein, kinesin, dynamin and the claret segregation protein.     

Poleward kinetochore fiber movement occurs during both metaphase and anaphase-A in newt lung cell mitosis

Microtubules in the mitotic spindles of newt lung cells were marked using local photoactivation of fluorescence. The movement of marked segments on kinetochore fibers was tracked by digital fluorescence microscopy in metaphase and anaphase and compared to the rate of chromosome movement. In metaphase, kinetochore oscillations toward and away from the poles were coupled to kinetochore fiber shortening and growth. Marked zones on the kinetochore microtubules, meanwhile, moved slowly polewards at a rate of approximately 0.5 micron/min, which identifies a slow polewards movement, or "flux," of kinetochore microtubules accompanied by depolymerization at the pole, as previously found in PtK2 cells (Mitchison, 1989b). Marks were never seen moving away from the pole, indicating that growth of the kinetochore microtubules occurs only at their kinetochore ends. In anaphase, marked zones on kinetochore microtubules also moved polewards, though at a rate slower than overall kinetochore-to-pole movement. Early in anaphase-A, microtubule depolymerization at kinetochores accounted on average for 75% of the rate of chromosome-to-pole movement, and depolymerization at the pole accounted for 25%. When chromosome-to-pole movement slowed in late anaphase, the contribution of depolymerization at the kinetochores lessened, and flux became the dominant component in some cells. Over the whole course of anaphase-A, depolymerization at kinetochores accounted on average for 63% of kinetochore fiber shortening, and flux for 37%. In some anaphase cells up to 45% of shortening resulted from the action of flux. We conclude that kinetochore microtubules change length predominantly through polymerization and depolymerization at the kinetochores during both metaphase and anaphase as the kinetochores move away from and towards the poles. Depolymerization, though not polymerization, also occurs at the pole during metaphase and anaphase, so that flux contributes to polewards chromosome movements throughout mitosis. Poleward force production for chromosome movements is thus likely to be generated by at least two distinct molecular mechanisms.     

XMAP215–EB1 Interaction Is Required for Proper Spindle Assembly and Chromosome Segregation in Xenopus Egg Extract

In metaphase Xenopus egg extracts, global microtubule growth is mainly promoted by two unrelated microtubule stabilizers, end-binding protein 1 (EB1) and XMAP215. Here, we explore their role and potential redundancy in the regulation of spindle assembly and function. We find that at physiological expression levels, both proteins are required for proper spindle architecture: Spindles assembled in the absence of EB1 or at decreased XMAP215 levels are short and frequently multipolar. Moreover, the reduced density of microtubules at the equator of ΔEB1 or ΔXMAP215 spindles leads to faulty kinetochore–microtubule attachments. These spindles also display diminished poleward flux rates and, upon anaphase induction, they neither segregate chromosomes nor reorganize into interphasic microtubule arrays. However, EB1 and XMAP215 nonredundantly regulate spindle assembly because an excess of XMAP215 can compensate for the absence of EB1, whereas the overexpression of EB1 cannot substitute for reduced XMAP215 levels. Our data indicate that EB1 could positively regulate XMAP215 by promoting its binding to the microtubules. Finally, we show that disruption of the mitosis-specific XMAP215–EB1 interaction produces a phenotype similar to that of either EB1 or XMAP215 depletion. Therefore, the XMAP215–EB1 interaction is required for proper spindle organization and chromosome segregation in Xenopus egg extracts.     

Micromanipulation of Chromosomes in Mitotic Vertebrate Tissue Cells: Tension Controls the State of Kinetochore Movement

In mitotic vertebrate tissue cells, chromosome congression to the spindle equator in prometaphase and segregation to the poles in anaphase depend on the movements of kinetochores at their kinetochore microtubule attachment sites. To test if kinetochores sense tension to control their states of movement poleward (P) and away from the pole (AP), we applied an external force to the spindle in preanaphase newt epithelial cells by stretching chromosome arms with microneedles. For monooriented chromosomes (only one kinetochore fiber), an abrupt stretch of an arm away from the attached pole induced the single attached kinetochore to persist in AP movement at about 2 μm/min velocity, resulting in chromosome movement away from the pole. When the stretch was reduced or the needle removed, the kinetochore switched to P movement at about 2 μm/min and pulled the chromosome back to near the premanipulation position within the spindle. For bioriented chromosomes (sister kinetochores attached to opposite poles) near the spindle equator, stretching one arm toward a pole placed the kinetochore facing away from the direction of stretch under tension and the sister facing toward the stretch under reduced tension or compression. Kinetochores under increased tension exhibited prolonged AP movement while kinetochores under reduced tension or compression exhibited prolonged P movement, moving the centromeres at about 2 μm/min velocities off the metaphase plate in the direction of stretch. Removing the needle resulted in centromere movement back to near the spindle equator at similar velocities. These results show that tension controls the direction of kinetochore movement and associated kinetochore microtubule assembly/disassembly to position centromeres within the spindle of vertebrate tissue cells. High tension induces persistent AP movement while low tension induces persistent P movement. The velocity of P and AP movement appears to be load independent and governed by the molecular mechanisms which attach kinetochores to the dynamic ends of kinetochore microtubules.     

Poleward microtubule flux is a major component of spindle dynamics and anaphase a in mitotic Drosophila embryos

During cell division, eukaryotic cells assemble dynamic microtubule-based spindles to segregate replicated chromosomes. Rapid spindle microtubule turnover, likely derived from dynamic instability, has been documented in yeasts, plants and vertebrates. Less studied is concerted spindle microtubule poleward translocation (flux) coupled to depolymerization at spindle poles. Microtubule flux has been observed only in vertebrates, although there is indirect evidence for it in insect spermatocytes and higher plants. Here we use fluorescent speckle microscopy (FSM) to demonstrate that mitotic spindles of syncytial Drosophila embryos exhibit poleward microtubule flux, indicating that flux is a widely conserved property of spindles. By simultaneously imaging chromosomes (or kinetochores) and flux, we provide evidence that flux is the dominant mechanism driving chromosome-to-pole movement (anaphase A) in these spindles. At 18 degrees C and 24 degrees C, separated sister chromatids moved poleward at average rates (3.6 and 6.6 microm/min, respectively) slightly greater than the mean rates of poleward flux (3.2 and 5.2 microm/min, respectively). However, at 24 degrees C the rate of kinetochore-to-pole movement varied from slower than to twice the mean rate of flux, suggesting that although flux is the dominant mechanism, kinetochore-associated microtubule depolymerization contributes to anaphase A.     

Sites of microtubule assembly and disassembly in the mitotic spindle

We have microinjected biotinylated tubulin into mitotic fibroblast cells to identify the sites in the spindle at which new subunits are incorporated into microtubules (MTs). Labeled subunits were visualized in the electron microscope using an antibody to biotin followed by a secondary antibody coupled to colloidal gold. Astral MTs incorporate labeled subunits very rapidly by elongation of existing MTs and by new nucleation from the centrosome. At a slower rate, kinetochore MTs incorporate subunits at the kinetochore progressively during metaphase, suggesting a slow poleward flux of subunits in the kinetochore fiber. When cells injected in metaphase were examined in anaphase, a significant fraction of kinetochore MTs was unlabeled, suggesting that depolymerization had occurred at the kinetochore concomitant with chromosome to pole movement. The existence of opposite fluxes at the kinetochore during metaphase and anaphase suggests that two separate forces are responsible for chromosome congression and anaphase movement.     

The Drosophila Kinesin-13, KLP59D,Impacts Pacman- and Flux-based Chromosome Movement

Chromosome movements are linked to the active depolymerization of spindle microtubule (MT) ends. Here we identify the kinesin-13 family member, KLP59D, as a novel and uniquely important regulator of spindle MT dynamics and chromosome motility in Drosophila somatic cells. During prometaphase and metaphase, depletion of KLP59D, which targets to centrosomes and outer kinetochores, suppresses the depolymerization of spindle pole–associated MT minus ends, thereby inhibiting poleward tubulin Flux. Subsequently, during anaphase, loss of KLP59D strongly attenuates chromatid-to-pole motion by suppressing the depolymerization of both minus and plus ends of kinetochore-associated MTs. The mechanism of KLP59D''s impact on spindle MT plus and minus ends appears to differ. Our data support a model in which KLP59D directly depolymerizes kinetochore-associated plus ends during anaphase, but influences minus ends indirectly by localizing the pole-associated MT depolymerase KLP10A. Finally, electron microscopy indicates that, unlike the other Drosophila kinesin-13s, KLP59D is largely incapable of oligomerizing into MT-associated rings in vitro, suggesting that such structures are not a requisite feature of kinetochore-based MT disassembly and chromosome movements.     

Kinetochore microtubule dynamics and the metaphase-anaphase transition

We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation. Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state. A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process. The movement of the activated zone was also measured along with chromosome movement and spindle elongation. To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase. We determined values for the turnover of kMTs (t1/2 = 7.1 +/- 2.4 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs. In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 37.5 +/- 8.5 min at 30 degrees C). Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value. In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16%. These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition. A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore. The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.     

Direct visualization of microtubule flux during metaphase and anaphase in crane-fly spermatocytes

Microtubule flux in spindles of insect spermatocytes, long-used models for studies on chromosome behavior during meiosis, was revealed after iontophoretic microinjection of rhodamine-conjugated (rh)-tubulin and fluorescent speckle microscopy. In time-lapse movies of crane-fly spermtocytes, fluorescent speckles generated when rh-tubulin incorporated at microtubule plus ends moved poleward through each half-spindle and then were lost from microtubule minus ends at the spindle poles. The average poleward velocity of approximately 0.7 microm/min for speckles within kinetochore microtubules at metaphase increased during anaphase to approximately 0.9 microm/min. Segregating half-bivalents had an average poleward velocity of approximately 0.5 microm/min, about half that of speckles within shortening kinetochore fibers. When injected during anaphase, rhtubulin was incorporated at kinetochores, and kinetochore fiber fluorescence spread poleward as anaphase progressed. The results show that tubulin subunits are added to the plus end of kinetochore microtubules and are removed from their minus ends at the poles, all while attached chromosomes move poleward during anaphase A. The results cannot be explained by a Pac-man model, in which 1) kinetochore-based, minus end-directed motors generate poleward forces for anaphase A and 2) kinetochore microtubules shorten at their plus ends. Rather, in these cells, kinetochore fiber shortening during anaphase A occurs exclusively at the minus ends of kinetochore microtubules.     

Hec1 and nuf2 are core components of the kinetochore outer plate essential for organizing microtubule attachment sites

A major goal in the study of vertebrate mitosis is to identify proteins that create the kinetochore-microtubule attachment site. Attachment sites within the kinetochore outer plate generate microtubule dependent forces for chromosome movement and regulate spindle checkpoint protein assembly at the kinetochore. The Ndc80 complex, comprised of Ndc80 (Hec1), Nuf2, Spc24, and Spc25, is essential for metaphase chromosome alignment and anaphase chromosome segregation. It has also been suggested to have roles in kinetochore microtubule formation, production of kinetochore tension, and the spindle checkpoint. Here we show that Nuf2 and Hec1 localize throughout the outer plate, and not the corona, of the vertebrate kinetochore. They are part of a stable "core" region whose assembly dynamics are distinct from other outer domain spindle checkpoint and motor proteins. Furthermore, Nuf2 and Hec1 are required for formation and/or maintenance of the outer plate structure itself. Fluorescence light microscopy, live cell imaging, and electron microscopy provide quantitative data demonstrating that Nuf2 and Hec1 are essential for normal kinetochore microtubule attachment. Our results indicate that Nuf2 and Hec1 are required for organization of stable microtubule plus-end binding sites in the outer plate that are needed for the sustained poleward forces required for biorientation at kinetochores.     

Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle

To test the popular but unproven assumption that the metaphase-anaphase transition in vertebrate somatic cells is subject to a checkpoint that monitors chromosome (i.e., kinetochore) attachment to the spindle, we filmed mitosis in 126 PtK1 cells. We found that the time from nuclear envelope breakdown to anaphase onset is linearly related (r2 = 0.85) to the duration the cell has unattached kinetochores, and that even a single unattached kinetochore delays anaphase onset. We also found that anaphase is initiated at a relatively constant 23-min average interval after the last kinetochore attaches, regardless of how long the cell possessed unattached kinetochores. From these results we conclude that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle. We also found that some cells treated with 0.3-0.75 nM Taxol, after the last kinetochore attached to the spindle, entered anaphase and completed normal poleward chromosome motion (anaphase A) up to 3 h after the treatment--well beyond the 9-48-min range exhibited by untreated cells. The fact that spindle bipolarity and the metaphase alignment of kinetochores are maintained in these cells, and that the chromosomes move poleward during anaphase, suggests that the checkpoint monitors more than just the attachment of microtubules at sister kinetochores or the metaphase alignment of chromosomes. Our data are most consistent with the hypothesis that the checkpoint monitors an increase in tension between kinetochores and their associated microtubules as biorientation occurs.     

Microtubules,chromosome movement,and reorientation after chromosomes are detached from the spindle by micromanipulation

The relationship between chromosome movement and mirotubules was explored by combining micromanipulation of living grasshopper spermatocytes with electron microscopy. We detached chromosomes from the spindle and placed them far out in the cytoplasm. Soon, the chromosomes began to move back toward the spindle and the cells were fixed at a chosen moment. The microtubules seen in three-dimensional reconstructions were correlated with the chromosome movement just prior to fixation. Before movement began, detached chromosomes had no kinetochore microtubules or a single one at most. Renewed movement was always accompanied by the reappearance of kinetochore microtubules; a single kinetochore microtubule appeared to suffice. Chromosome movements and kinetochore microtubule arrangements were unusual after reattachment, but their relationship was not: poleward forces, parallel to the kinetochore microtubule axis (as in normal anaphase), would explain the movement, however odd. The initial arrangement of kinetochore microtubules would have led to aberrant chromosome distribution if it persisted, but instead, reorientation to the appropriate arrangement always followed. Observations on living cells permitted us to place in sequence the kinetochore microtubule arrangements seen in fixed cells, revealing the microtubule transformations during reorientation. From the sequence of events we conclude that chromosome movement can cause reorientation to begin and that in the changes which follow, an unstable attachment of kinetochore microtubules to the spindle plays a major role.     

Mitotic motors and chromosome segregation: the mechanism of anaphase B

Anaphase B spindle elongation plays an important role in chromosome segregation. In the present paper, we discuss our model for anaphase B in Drosophila syncytial embryos, in which spindle elongation depends on an ip (interpolar) MT (microtubule) sliding filament mechanism generated by homotetrameric kinesin-5 motors acting in concert with poleward ipMT flux, which acts as an 'on/off' switch. Specifically, the pre-anaphase B spindle is maintained at a steady-state length by the balance between ipMT sliding and ipMT depolymerization at spindle poles, producing poleward flux. Cyclin B degradation at anaphase B onset triggers: (i) an MT catastrophe gradient causing ipMT plus ends to invade the overlap zone where ipMT sliding forces are generated; and (ii) the inhibition of ipMT minus-end depolymerization so flux is turned 'off', tipping the balance of forces to allow outward ipMT sliding to push apart the spindle poles. We briefly comment on the relationship of this model to anaphase B in other systems.     

The microtubule cross-linker Feo controls the midzone stability,motor composition,and elongation of the anaphase B spindle in Drosophila embryos

Chromosome segregation during anaphase depends on chromosome-to-pole motility and pole-to-pole separation. We propose that in Drosophila embryos, the latter process (anaphase B) depends on a persistent kinesin-5–generated interpolar (ip) microtubule (MT) sliding filament mechanism that “engages” to push apart the spindle poles when poleward flux is turned off. Here we investigated the contribution of the midzonal, antiparallel MT-cross-linking nonmotor MAP, Feo, to this “slide-and-flux-or-elongate” mechanism. Whereas Feo homologues in other systems enhance the midzone localization of the MT-MT cross-linking motors kinesin-4, -5 and -6, the midzone localization of these motors is respectively enhanced, reduced, and unaffected by Feo. Strikingly, kinesin-5 localizes all along ipMTs of the anaphase B spindle in the presence of Feo, including at the midzone, but the antibody-induced dissociation of Feo increases kinesin-5 association with the midzone, which becomes abnormally narrow, leading to impaired anaphase B and incomplete chromosome segregation. Thus, although Feo and kinesin-5 both preferentially cross-link MTs into antiparallel polarity patterns, kinesin-5 cannot substitute for loss of Feo function. We propose that Feo controls the organization, stability, and motor composition of antiparallel ipMTs at the midzone, thereby facilitating the kinesin-5–driven sliding filament mechanism underlying proper anaphase B spindle elongation and chromosome segregation.     

WDR62 localizes katanin at spindle poles to ensure synchronous chromosome segregation

Mutations in the WDR62 gene cause primary microcephaly, a pathological condition often associated with defective cell division that results in severe brain developmental defects. The precise function and localization of WDR62 within the mitotic spindle is, however, still under debate, as it has been proposed to act either at centrosomes or on the mitotic spindle. Here we explored the cellular functions of WDR62 in human epithelial cell lines using both short-term siRNA protein depletions and long-term CRISPR/Cas9 gene knockouts. We demonstrate that WDR62 localizes at spindle poles, promoting the recruitment of the microtubule-severing enzyme katanin. Depletion or loss of WDR62 stabilizes spindle microtubules due to insufficient microtubule minus-end depolymerization but does not affect plus-end microtubule dynamics. During chromosome segregation, WDR62 and katanin promote efficient poleward microtubule flux and favor the synchronicity of poleward movements in anaphase to prevent lagging chromosomes. We speculate that these lagging chromosomes might be linked to developmental defects in primary microcephaly.     

Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells

During mitosis in cultured newt pneumocytes, one or more chromosomes may become positioned well removed (greater than 50 microns) from the polar regions during early prometaphase. As a result, these chromosomes are delayed for up to 5 h in forming an attachment to the spindle. The spatial separation of these chromosomes from the polar microtubule-nucleating centers provides a unique opportunity to study the initial stages of kinetochore fiber formation in living cells. Time-lapse Nomarski-differential interference contrast videomicroscopic observations reveal that late-attaching chromosomes always move, upon attachment, into a single polar region (usually the one closest to the chromosome). During this attachment, the kinetochore region of the chromosome undergoes a variable number of transient poleward tugs that are followed, shortly thereafter, by rapid movement of the chromosome towards the pole. Anti-tubulin immunofluorescence and serial section EM reveal that the kinetochores and kinetochore regions of nonattached chromosomes lack associated microtubules. By contrast, these methods reveal that the attachment and subsequent poleward movement of a chromosome correlates with the association of a single long microtubule with one of the kinetochores of the chromosome. This microtubule traverses the entire distance between the spindle pole and the kinetochore and often extends well past the kinetochore. From these results, we conclude that the initial attachment of a chromosome to the newt pneumocyte spindle results from an interaction between a single polar-nucleated microtubule and one of the kinetochores on the chromosome. Once this association is established, the kinetochore is rapidly transported poleward along the surface of the microtubule by a mechanism that is not dependent on microtubule depolymerization. Our results further demonstrate that the motors for prometaphase chromosome movement must be either on the surface of the kinetochore (i.e., within the corona but not the plate), distributed along the surface of the kinetochore microtubules, or both.     

Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 microM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2--5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.